Outer hair cells (OHCs) in the mammalian cochlea are inhibited by the release of acetylcholine (ACh) from efferent neurons. An analogous inhibitory mechanism is found in all vertebrate inner ears. Inhibition results from the activation of small-conductance calcium-dependent (SK) potassium channels. The hair cell's ACh receptor (AChR) is a ligand-gated cation channel whose nicotinic subunits, alpha9 and alpha10, have significant calcium permeability in functional expression studies. Thus, a compelling argument exists that calcium influx through the hair cell AChR directly activates SK channels. An unresolved question is whether the nearby synaptic cistern might release additional calcium. If so, under what conditions does calcium release play a significant role? What molecular mechanisms couple the AChR to the synaptic cistem? In this proposal we will employ biophysical and molecular genetic approaches to tackle this question in both avian and mammalian hair cells. Pharmacological agents that alter calcium store function will be applied during voltage-clamp recording from hair cells (with control experiments on alpha9/alpha10 expressed in oocytes). These treatments will be used during application of ACh to isolated hair cells, or while stimulating ACh release from efferent synaptic terminals in an excised organ of Corti preparation. Similar studies will be conducted on hair cells of the crooked neck dwarf chicken that lacks functional type 1 Ryan dine receptors. Transgenic mice will be generated and tested to probe AChR coupling to downstream signals. How is inhibition affected when so-called 'gain of function' AChRs are present? Does excess calcium influx through such 'super receptors' alter presumptive store-dependent components of the cholinergic response? In contrast, how is efferent input and the cholinergic response altered when hair cell AChRs are calcium irnpermeant? If inhibition persists without calcium influx, then other signaling, through conformational coupling or the generation of IP3, must be investigated. Emerging evidence suggests that ryanodine- and IP3-sensitive stores may predominate in hair cells from different regions of the cochlea. Finally, as our studies on calcium stores progress, new transgenic manipulations will be designed to directly test emerging hypotheses of signaling within the hair cell. These studies will add to our basic knowledge of calcium metabolism in hair cells. Calcium excitotoxicity could be instrumental in hair cell loss following trauma and throughout presbycusis.